Project Summary: Cardiovascular disease is the leading cause of death and disability worldwide. Due to the limited regenerative capacity of adult hearts, human induced pluripotent stem cells (hiPSCs) have received significant attention due to their proven ability to derive functional cardiomyocytes (hiPSC-CMs). Despite the progress, the current inability to effectively deliver and integrate hiPSC-CMs into damaged myocardium has limited the applications of hiPSC technology in cardiac repair. The current strategies are limited by 1) low cell retention and survival after cell transplantation, and 2) unspecific and immature phenotype of the current hiPSC-CMs. To address these challenges, we pioneered the use of electrically conductive silicon nanowires (e-SiNWs) to facilitate self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac spheroids. The addition of e-SiNWs was found to enhance electrical conduction in the spheroids and improve their function. In addition, our recent publication showed electrical stimulation synergizes with e-SiNWs to promote ventricular lineage specification and cellular maturation (i.e., ventricular maturation) and reduce the spontaneous beating of the hiPSC cardiac spheroids in in vitro culture. Further, our in vivo studies showed the nanowired spheroids improve cell retention and engraftment with host myocardium after transplantation, presumably due to their 3D microtissue configuration and the e-SiNW enhanced electrical integration. Our long-term goal is to translate nanowired hiPSC cardiac spheroid technology into a clinical therapy for heart repair. The goal of this proposal is to 1) study the effects of the intrinsic (electrical and surface) properties of e-SiNWs and extrinsic factor (electrical stimulation) on ventricular maturation of the nanowired spheroids, and 2) examine the effects of ventricular maturity of the nanowired spheroids on their engraftment. The central hypothesis of this proposal is the nanowired hiPSC cardiac spheroids provide a powerful platform to 1) accelerate ventricular maturation of hiPSC-CMs in vitro and 2) improve the retention, engraftment and integration of hiPSC-CMs with host myocardium in vivo. The proposal is innovative in that, for the first time, we prepare hiPSC cardiac microtissues with defined ventricular maturity for transplantation. Accordingly, we will pursue three specific aims: 1) Elucidate the mechanisms of the interactions between e-SiNWs and hiPSC-CMs, 2) Further advance ventricular maturation of nanowired hiPSC cardiac spheroids through long-term electrical stimulations, and 3) Examine in vivo efficacy of the nanowired hiPSC cardiac spheroids in both healthy (Aim 3a) and injured (Aim 3b) rat hearts. The completion of the proposed research would, for the first time, allow us to 1) produce hiPSC-CMs with controlled ventricular maturity, 2) develop a set of quantitative criteria to assess ventricular maturity of hiPSC-CMs for transplantation, and 3) identify a suitable range of ventricular maturity of hiPSC- CMs for transplantation. Also, it will lay down a solid foundation to establish the use of electrically stimulated, nanowired hiPSC cardiac spheroids as an innovative platform to deliver hiPSC-CMs to treat heart failure.